Screening Of Germplasm Resources And Inter-rhizosphere Soil Microbial Research In Atractylodes Chinensis

Atractylodis Rhizoma is derived from the dried rhizomes of Atractylodes lancea（Thunb.）DC.or Atractylodes chinensis（DC.）Koidz.It is clinically effective in strengthening the spleen,drying dampness and dispelling wind and cold.In recent years,the market demand for A.chinensis has been high and the supply has exceeded the demand,which has made it inevitable for A.chinensis to become a domestic species.Our group found after preliminary research that the phenotypic variation of A.chinensis plant after wild to domestic species is large and the quality of herbs is unstable,which seriously affects the clinical efficacy,so the selection and breeding of good strains with stable traits of A.chinensis is more urgent.In the cultivation process of A.chinensis,root rot is the biggest constraint to yield and quality,and the variation of soil microorganisms directly affects the occurrence of root rot.In this paper,we studied the selection of good germplasm resources and soil microorganisms between the roots of A.chinensis to provide a basis for the selection and breeding of good strains of A.chinensis and a theoretical basis for solving the root rot disease of A.chinensis.Objective:To determine the suitable distribution areas of wild resources of A.chinensis;to establish a germplasm resource bed for A.chinensis;to screen high-quality germplasm resources;to establish a detection method based on high-performance liquid chromatography（HPLC）for simultaneous detection of both components of A.chinensis and to evaluate the agronomic indexes,active ingredients and seed quality of the selected germplasm resources.To analyze the diversity of soil microorganisms in A.chinensis and to provide a theoretical basis for exploring and solving the pathogenesis and control of root rot of A.chinensisMethods:1.We collected wild germplasm resources of A.chinensis in Hebei Province and investigated the information of A.chinensis resources in cultivated areas in the field to determine the range of A.chinensis distribution area.We collected live seed roots from the distribution area and planted them in the resource garden to establish a germplasm resource garden,protected the germplasm,and harvested seeds for sowing in the selection field.The growth traits of A.chinensis were consistent and single plants were listed,and observation records were made from the growth consistency,leaf morphology,rhizome other appearance shapes to make preliminary screening of excellent germplasm strains.2.During each growth period of the preliminary selection,field growth traits were observed and recorded every 20 d.Chlorophyll and photosynthetic rate contents were measured,together with malondialdehyde（MDA）content,superoxide dismutase（SOD）and peroxidase（POD）enzyme activities of fresh rhizomes of A.chinensis and soil sucrase（S-SC）,soil neutral phosphatase（S-NP）and soil alkaline phosphatase（S-NP）of inter-rhizosphere soil.Soil alkaline phosphatase（S-AKP）,soil cellulase（S-CL）enzyme activities in the inter-root soil were analyzed to evaluate the potential selection advantages of the five lines.3.High-performance liquid chromatography（HPLC）was used to establish a method for the determination of the content of the dual components（atractylenol and atractylenone）of A.chinensis herbs,and a methodological investigation was conducted,together with moisture and ash tests to evaluate the quality of the collected germplasm resources and the primary A.chinensis herbs.Ten plants of each strain of A.chinensis were selected,the fresh roots were harvested,and the dry and fresh weights of individual roots were determined separately.SPSS22.0 and Origin2021 software were used for correlation analysis and quality evaluation of the data on neatness,thousand-grain weight and germination rate of the primed A.chinensis seeds.4.Using Illumina Hi Seq high-throughput sequencing technology,the diversity of soil bacterial and fungal communities in the inter-rhizosphere of root-rotting A.chinensis and healthy plants were comparatively analyzed,and soil physicochemical properties and enzyme activities were analyzed.Results:1.Based on field surveys in the distribution areas and cultivation sites of wild resources of A.chinensis,a total of 406 pieces of information on the distribution of A.chinensis were collected to determine the distribution area range of A.chinensis in the distribution area concentration points of Chengde,Zhangjiakou and Qinhuangdao.A total of 59 living seed roots of A.chinensis were collected and planted in the resource bed to establish the A.chinensis germplasm resource bed.Preliminary screening of five strains of A.chinensis excellent germplasm resources:A.chinensis No.1（BCZ1）,A.chinensis No.2（BCZ2）,A.chinensis No.3（BCZ3）,A.chinensis No.4（BCZ4）,A.chinensis No.5（BCZ5）.2.The best growth temperature during the seedling stage was 20～24℃,and the peak growth of BCZ4 was from mid-May to early August.The plant height,chlorophyll content,and photosynthetic rate of BCZ4 were the highest among all lines,and from the beginning of September,the leaves of each line gradually withered and the plants started to age.The fresh root enzyme activity of BCZ4 had the lowest malondialdehyde（MDA）content,the highest superoxide dismutase（SOD）and peroxidase（POD）enzyme activities,and the lowest POD activity of BCZ2.The soil enzyme activities of each strain were determined as follows:soil neutral phosphatase,soil cellulase,and soil alkaline phosphatase,and soil sucrase activities were the highest in BCZ3 and relatively low in BCZ4.3.A method based on high performance liquid chromatography（HPLC）was developed for the simultaneous determination of atractylenol and atractylone in A.chinensis herb using a Discovery（?）C18 column（4.6 mm×250mm,5μm）with a gradient elution of 0.2%phosphate water（A）-acetonitrile（B）as the mobile phase,the detection wavelength was 220 nm,the flow rate was1.0 m L·min^-1 The linearity was y=10.543x+0.1889（R2=0.9999）with an average recovery of 105.20%for atractylenol and y=1.615x-0.007（R²=0.9975）with an average recovery of 92.72%for atractylenone.The 35cultivated herbs from the germplasm resource nursery were determined to contain 0.02%～0.84%of atractylenol and 0.06%～1.74%of atractylenone.The35 samples collected had a high qualification rate;the quality of the five strains of Cangjiao herbs screened were all qualified.The dried and fresh seeds of the five lines of Atractylodes were BCZ3>BCZ2>BCZ4>BCZ1>BCZ5>CK.The BCZ3 line improved 54.86%compared with the control group.In the quality evaluation of the seeds of the five lines of C.nigra,the seed width was2.6～2.17 mm.seed thickness was 1.29～1.75 mm.germination rate from high to low was:BCZ3>BCZ2=BCZ5>BCZ1>BCZ4,the maximum was 86%and the minimum was 49%.4.The pH of inter-rhizosphere soil of A.chinensis（5.1）was lower than that of healthy soil（5.9）,and other physicochemical indexes of soil and soil enzyme activity content were significantly higher than that of healthy soil;the activities of fast-acting N,fast-acting P,fast-acting K,and soil neutral phosphatase,soil alkaline phosphatase,soil urease,soil cellulase,and soil sucrase of root rot soil were higher than those of healthy soil,and there were significant differences.Analysis of inter-root microbial diversity:At the level of bacterial phylum classification,the inter-root soils of A.chinensis with root rot had Actinobacteriota,Chloroflexi,Gemmatimonadota,Myxococcota,The abundance of Methylomirabilota,Nitrospirota was lower than that of healthy inter-rhizosphere soil;the abundance of Proteobacteria,Acidobacteriota,Firmicutes,Bacteroidota in root rot soil was lower than that of healthy inter-rhizosphere soil.The abundance of Proteobacteria,Acidobacteriota,Firmicutes,and Bacteroidota was higher than that of healthy soil;the abundance of Pseudomonadales in root rot soil samples was 6.34%on average,while it was not found in healthy soil samples.At the level of fungal phylum classification,the abundance of Ascomycetes in root rot soil was higher than that in healthy soil,and the abundance of Peridiomycetes and Stenotrophomonas was lower than that in healthy soil;at the level of fungal genus classification,the abundance of Nectriaceae and Fusarium in root rot soil was higher than that in healthy soil,and the abundance of Cheatomium in root rot soil was The abundance of Cheatomium in root rot soil was 6.83%lower than that in healthy soil.Conclusion:1.The concentrated distribution areas of wild and domestic A.chinensis in Hebei Province are:Chengde,Qinhuangdao,and Zhangjiakou;59 A.chinensis germplasm resources（24 wild and 35 cultivated）were collected in this area,and an A.chinensis germplasm resource bed was established to preserve the living A.chinensis germplasm in situ.Preliminarily,five good strains of A.chinensis were selected:BCZ1 for flower color variant;BCZ2 for leaf re-divided type;BCZ3 for main stem sturdy total inflorescence fan-shaped;BCZ4 for leaf lower less divided type;BCZ5 for plant branching in clusters.2.The agronomic indexes of the five strains of A.chinensis showed that BCZ4 was significantly different from the other strains.In the assay of enzyme activity content of fresh herbs,SOD and POD,the main indicators of resistance,were highest in BCZ4,followed by BCZ3,and the lowest MDA content was found in BCZ4.The inter-root soil is involved in various physiological reactions of plant growth,and it was found that the inter-root soil enzyme activities S-NP,S-SC,S-CL,and S-AKP were the highest in BCZ3.It was tentatively inferred that BCZ4 and BCZ3 were more resistant to stress.3.A method based on HPLC was established for the determination of the contents of Atractylodin and Atractylon in A.chinensis herbs,which is reproducible and accurate and can be used for the quality evaluation of A.chinensis herbs.There were significant differences in the quality of different growth years of A.chinensis and the herb quality and seed quality of the five strains of A.chinensis screened were qualified.The five preliminarily screened lines of A.chinensis were of qualified herb quality and good quality,BCZ4 had the highest atractylenin content,and BCZ3 had the highest atractylenone content with obvious yield growth,which could be selected according to different needs.In the seed evaluation of the five lines,BCZ2 has the highest thousand grain weight and BCZ3 has the highest germination rate,which has the potential of seed type selection.4.Experiments on the physicochemical properties of inter-rhizosphere soil and healthy inter-rhizosphere soil with root rot disease A.chinensis yielded that the p H of root rot soil was lower than that of healthy soil.In the analysis of microbial diversity in the inter-rhizosphere soil of C.nigra plants,the bacterial diversity in the root rot soil was higher than that in the healthy soil,and the abundance of Actinomycetes,as common soil-borne disease antagonists,was less in the root rot soil than in the healthy soil.Fungal abundance was lower than that of healthy soils.Fusarium could promote the increase of pathogenic bacteria,and the abundance of Fusarium was higher in root rot soils than in healthy soils.As a common biocontrol fungus,the abundance of the genus Cheatomium is significantly reduced in the root rot soils of the A.chinensis than in healthy soils.Therefore,it was tentatively determined that the occurrence of root rot disease in northern celandine was associated with soil enzyme activity,soil physicochemical properties,and microbial diversity.